Combining fibril-induced alpha-synuclein aggregation and 6-hydroxydopamine in a mouse model of Parkinson's disease and the effect of cerebral dopamine neurotrophic factor on the induced neurodegeneration.

The existent pre-clinical models of Parkinson's disease do not simultaneously recapitulate severe degeneration of dopamine neurons and the occurrence of alpha-synuclein (aSyn) aggregation in one study system. In this study, we injected aSyn pre-formed fibrils (PFF) and 6-hydroxydopamine (6-OHDA) unilaterally into the striatum of C57BL/6 wild-type male mice at an interval of 2 weeks to induce aggregation of aSyn protein and trigger the loss of dopamine neurons simultaneously in one model and studied the behavioural effects of the combination in these mice. 6-OHDA was tested at three different doses, and 2 µg of 6-OHDA combined with PFF-induced aSyn aggregation was found to produce the most optimal disease phenotype. At 14 weeks timepoint, mice injected with a combination of PFF and 6-OHDA sustained significant damage to the nigrostriatal pathway and exhibited aSyn-positive aggregation. Our data suggest that the neurons that formed large aSyn aggregates were particularly vulnerable to 6-OHDA-induced degeneration. We also demonstrate the manifestation of a relatively aggressive pathology in 2- to 4-month-old mice, as compared to younger 7- to 9-week-old ones. Furthermore, cerebral dopamine neurotrophic factor (CDNF) administered intrastriatally rescued dopamine neurons and motor behaviour of the animals to some extent from 6-OHDA toxicity. However, no such effect could be seen in the novel 6-OHDA + PFFs combination model. For the first time, we demonstrate the combined effect of PFF and 6-OHDA simultaneously in one model. We further discuss the scope for further optimizing this combination model to develop it as a promising pre-clinical platform for drug screening and development.

Feasibility of combining alpha-synuclein aggregation and 6-OHDA in embryonic midbrain culture for modeling dopamine neuron degeneration.

Parkinson's disease (PD) is characterized by the loss of nigrostriatal dopamine (DA) neurons and the presence of alpha-synuclein (αSyn)-positive Lewy body (LB) pathology. In this study, we attempted to recapitulate both these features in a novel in vitro model for PD. To achieve this, we combined the αSyn pre-formed fibril (PFF)-seeded LB-like pathology with 6-hydroxydopamine (6-OHDA)-induced mitochondrial toxicity in mouse embryonic midbrain cultures. To pilot the model for therapeutics testing, we assessed the effects of cerebral dopamine neurotrophic factor (CDNF) on αSyn aggregation and neuron survival. PFF-seeded pathology did not lead to DA neuron loss even with the highest dose of PFFs. The combination of PFFs and 6-OHDA did not trigger additional neurodegeneration or LB-like pathology and instead presented DA neuron loss to a similar extent as with 6-OHDA only. CDNF did not affect the PFF-seeded αSyn pathology or the DA neuron survival in the combination model but showed a trend toward neuroprotection in the 6-OHDA-only cultures.

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia.

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A2AR), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner.

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo.

A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.

Soluble CD163 Changes Indicate Monocyte Association With Cognitive Deficits in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder with a significant immune component, as demonstrated by changes in immune biomarkers in patients' biofluids. However, which specific cells are responsible for those changes is unclear because most immune biomarkers can be produced by various cell types. OBJECTIVES: The aim of this study was to explore monocyte involvement in PD. METHODS: We investigated the monocyte-specific biomarker sCD163, the soluble form of the receptor CD163, in cerebrospinal fluid (CSF) and serum in two experiments, and compared it with other biomarkers and clinical data. Potential connections between CD163 and alpha-synuclein were studied in vitro. RESULTS: CSF-sCD163 increased in late-stage PD and correlated with the PD biomarkers alpha-synuclein, Tau, and phosphorylated Tau, whereas it inversely correlated with the patients' cognitive scores, supporting monocyte involvement in neurodegeneration and cognition in PD. Serum-sCD163 increased only in female patients, suggesting a sex-distinctive monocyte response. CSF-sCD163 also correlated with molecules associated with adaptive and innate immune system activation and with immune cell recruitment to the brain. Serum-sCD163 correlated with proinflammatory cytokines and acute-phase proteins, suggesting a relation to chronic systemic inflammation. Our in vitro study showed that alpha-synuclein activates macrophages and induces shedding of sCD163, which in turn enhances alpha-synuclein uptake by myeloid cells, potentially participating in its clearance. CONCLUSIONS: Our data present sCD163 as a potential cognition-related biomarker in PD and suggest a role for monocytes in both peripheral and brain immune responses. This may be directly related to alpha-synuclein's proinflammatory capacity but could also have consequences for alpha-synuclein processing. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

ADHD-like behaviors caused by inactivation of a transcription factor controlling the balance of inhibitory and excitatory neuron development in the mouse anterior brainstem.

The neural circuits regulating motivation and movement include midbrain dopaminergic neurons and associated inhibitory GABAergic and excitatory glutamatergic neurons in the anterior brainstem. Differentiation of specific subtypes of GABAergic and glutamatergic neurons in the mouse embryonic brainstem is controlled by a transcription factor Tal1. This study characterizes the behavioral and neurochemical changes caused by the absence of Tal1 function. The Tal1cko mutant mice are hyperactive, impulsive, hypersensitive to reward, have learning deficits and a habituation defect in a novel environment. Only minor changes in their dopaminergic system were detected. Amphetamine induced striatal dopamine release and amphetamine induced place preference were normal in Tal1cko mice. Increased dopamine signaling failed to stimulate the locomotor activity of the Tal1cko mice, but instead alleviated their hyperactivity. Altogether, the Tal1cko mice recapitulate many features of the attention and hyperactivity disorders, suggesting a role for Tal1 regulated developmental pathways and neural structures in the control of motivation and movement.

GDNF/RET Signaling Pathway Activation Eliminates Lewy Body Pathology in Midbrain Dopamine Neurons.

BACKGROUND: Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded α-synuclein (α-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded α-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. OBJECTIVES: We studied the effects of GDNF on misfolded α-syn accumulation in DA neurons. METHODS: Lewy pathology progression was modeled by the application of α-syn preformed fibrils in cultured DA neurons and in the adult mice. RESULTS: We discovered that GDNF prevented accumulation of misfolded α-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced α-syn accumulation. CONCLUSIONS: For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded α-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. © 2020 International Parkinson and Movement Disorder Society.

Cerebral dopamine neurotrophic factor-deficiency leads to degeneration of enteric neurons and altered brain dopamine neuronal function in mice.

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf-/-), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf-/- mouse are much greater than in those of Cdnf+/+ mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf-/- mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf-/- male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf-/- mouse brain is altered. The deficiencies of Cdnf-/- mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.

Conditioned Aversion and Neuroplasticity Induced by a Superagonist of Extrasynaptic GABAA Receptors: Correlation With Activation of the Oval BNST Neurons and CRF Mechanisms.

THIP (gaboxadol), a superagonist of the δ subunit-containing extrasynaptic GABAA receptors, produces persistent neuroplasticity in dopamine (DA) neurons of the ventral tegmental area (VTA), similarly to rewarding drugs of abuse. However, unlike them THIP lacks abuse potential and induces conditioned place aversion in mice. The mechanism underlying the aversive effects of THIP remains elusive. Here, we show that mild aversive effects of THIP were detected 2 h after administration likely reflecting an anxiety-like state with increased corticosterone release and with central recruitment of corticotropin-releasing factor corticotropin-releasing factor receptor 1 (CRF1) receptors. A detailed immunohistochemical c-Fos expression mapping for THIP-activated brain areas revealed a correlation between the activation of CRF-expressing neurons in the oval nucleus of the bed nuclei of stria terminalis and THIP-induced aversive effects. In addition, the neuroplasticity of mesolimbic DA system (24 h after administration) and conditioned place aversion by THIP after four daily acute sessions were dependent on extrasynaptic GABAA receptors (abolished in δ-GABAA receptor knockout mice) and activation of the CRF1 receptors (abolished in wildtype mice by a CRF1 receptor antagonist). A selective THIP-induced activation of CRF-expressing neurons in the oval part of the bed nucleus of stria terminalis may constitute a novel mechanism for inducing plasticity in a population of VTA DA neurons and aversive behavioral states.

Prolyl Oligopeptidase Regulates Dopamine Transporter Phosphorylation in the Nigrostriatal Pathway of Mouse.

Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson's disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the α-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of α-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.

Dampened Amphetamine-Stimulated Behavior and Altered Dopamine Transporter Function in the Absence of Brain GDNF.

Midbrain dopamine neuron dysfunction contributes to various psychiatric and neurological diseases, including drug addiction and Parkinson's disease. Because of its well established dopaminotrophic effects, the therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) has been studied extensively in various disorders with disturbed dopamine homeostasis. However, the outcomes from preclinical and clinical studies vary, highlighting a need for a better understanding of the physiological role of GDNF on striatal dopaminergic function. Nevertheless, the current lack of appropriate animal models has limited this understanding. Therefore, we have generated novel mouse models to study conditional Gdnf deletion in the CNS during embryonic development and reduction of striatal GDNF levels in adult mice via AAV-Cre delivery. We found that both of these mice have reduced amphetamine-induced locomotor response and striatal dopamine efflux. Embryonic GDNF deletion in the CNS did not affect striatal dopamine levels or dopamine release, but dopamine reuptake was increased due to increased levels of both total and synaptic membrane-associated dopamine transporters. Collectively, these results suggest that endogenous GDNF plays an important role in regulating the function of dopamine transporters in the striatum.SIGNIFICANCE STATEMENT Delivery of ectopic glial cell line-derived neurotrophic factor (GDNF) promotes the function, plasticity, and survival of midbrain dopaminergic neurons, the dysfunction of which contributes to various neurological and psychiatric diseases. However, how the deletion or reduction of GDNF in the CNS affects the function of dopaminergic neurons has remained unknown. Using conditional Gdnf knock-out mice, we found that endogenous GDNF affects striatal dopamine homeostasis and regulates amphetamine-induced behaviors by regulating the level and function of dopamine transporters. These data regarding the physiological role of GDNF are relevant in the context of neurological and neurodegenerative diseases that involve changes in dopamine transporter function.

Correction: GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

[This corrects the article DOI: 10.1371/journal.pgen.1005710.].

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson's disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson's disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3'UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson's disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3'UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3'UTR targeting may constitute a useful tool in analyzing gene function.

Changes in neuronal dopamine homeostasis following 1-methyl-4-phenylpyridinium (MPP+) exposure.

1-Methyl-4-phenylpyridinium (MPP(+)), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP(+) exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DAcyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP(+) exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP(+) concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP(+) depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DAcyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP(+)-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DAcyt levels in MPP(+)-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP(+)-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP(+) on neuronal DA homeostasis and neurotoxicity.

GABAA receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area.

GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids) have been found to induce plasticity in the ventral tegmental area (VTA) dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of the circuitry.

Importance of GluA1 subunit-containing AMPA glutamate receptors for morphine state-dependency.

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional µ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

GABA site agonist gaboxadol induces addiction-predicting persistent changes in ventral tegmental area dopamine neurons but is not rewarding in mice or baboons.

Dopamine neurons of the ventral tegmental area (VTA) are involved at early phases of drug addiction. Even the first in vivo dose of various abused drugs induces glutamate receptor plasticity at the excitatory synapses of these neurons. Benzodiazepines that suppress the inhibitory GABAergic interneurons in the VTA via facilitation of synaptic GABA(A) receptors have induced neuroplasticity in dopamine neurons due to this disinhibitory mechanism. Here, we have tested a non-benzodiazepine direct GABA site agonist 4,5,6,7-tetrahydroisoxazolol[4,5-c]pyridine-3-ol (THIP) (also known as gaboxadol) that acts preferentially via high-affinity extrasynaptic GABA(A) receptors. A single sedative dose of THIP (6 mg/kg) to mice induced glutamate receptor plasticity for at least 6 d after administration. Increased AMPA/NMDA receptor current ratio and increased frequency, amplitude, and rectification of AMPA receptor responses suggested persistent targeting of GluA2-lacking AMPA receptors in excitatory synapses of VTA dopamine neurons ex vivo after THIP administration. This effect was abolished in GABA(A) receptor δ(-/-) mice, which have a loss of extrasynaptic GABA(A) receptors. In behavioral experiments, we found neither acute reinforcement in intravenous self-administration sessions with THIP at relevant doses using a yoked control paradigm in mice nor in baboons using a standard paradigm for assessing drug abuse liability; nor was any place preference found after conditioning sessions with various doses of THIP but rather a persistent aversion in 6 mg/kg THIP-conditioned mice. In summary, we found that activation of extrasynaptic δ-subunit-containing GABA(A) receptors leads to glutamate receptor plasticity of VTA dopamine neurons, but is not rewarding, and, instead, induces aversion.

Evidence for a role of inhibition of orexinergic neurons in the anxiolytic and sedative effects of diazepam: A c-Fos study.

The classical benzodiazepine diazepam (DZ) induces anxiolysis at low doses and sedation and hypnosis at higher doses. Different brain areas and neuronal populations most likely mediate these different behavioral effects. We used c-Fos immunohistochemistry as an indirect way to study neuronal activation or inhibition induced by DZ at anxiolytic and sedative doses (0.5 and 5mg/kg, respectively) in various brain areas involved in anxiety, arousal, sedation and addiction in C57BL/6J mice. We also focused on the two neuronal populations, orexinergic and dopaminergic neuronal populations, with the help of double-immunohistochemistry using c-Fos and orexin-A antibodies and c-Fos and tyrosine hydroxylase antibodies. We found that different brain areas of unhabituated mice reacted differently to the mild stress induced by vehicle injection. Also the response to anxiolytic or sedative doses of DZ differed between the areas, suggesting that distinct brain areas mediate the behavioral effects of low and high DZ doses. Our findings propose a role for inhibition of orexin neurons in the anxiolytic and sleep-promoting effects of DZ. In addition, the activation of central amygdala neurons by DZ treatment was associated with anxiolytic and sedative effects. On the other hand, the ventral hippocampus, basolateral amygdala, ventral tegmental area and prefrontal cortex were sensitive even to the mild injection stress, but not to the anxiolytic dose of DZ.

Excessive novelty-induced c-Fos expression and altered neurogenesis in the hippocampus of GluA1 knockout mice.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 subunit-deficient (GluA1-/-) mice display novelty-induced hyperactivity, cognitive and social defects and may model psychiatric disorders, such as schizophrenia and depression/mania. We used c-Fos expression in GluA1-/- mice to identify brain regions responsible for novelty-induced hyperlocomotion. Exposure to a novel cage for 2 h significantly increased c-Fos expression in many brain regions in both wild-type and knockout mice. Interestingly, the clearest genotype effect was observed in the hippocampus and its main input region, the entorhinal cortex, where the novelty-induced c-Fos expression was more strongly enhanced in GluA1-/- mice. Their novelty-induced hyperlocomotion partly depended on the activity of AMPA receptors, as it was diminished by the AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX) and unaffected by the AMPA receptor potentiator 2,3-dihydro-1,4-benzodioxin-6-yl-1-piperidinylmethanone (CX546). The hyperlocomotion of GluA1-/- mice was normalised to the level of wild-type mice within 5-6 h, after which their locomotion followed normal circadian rhythm and was not affected by acute or chronic treatments with the selective serotonin reuptake inhibitor escitalopram. We propose that hippocampal dysfunction, as evidenced by the excessive c-Fos response to novelty, is the major contributor to novelty-induced hyperlocomotion in GluA1-/- mice. Hippocampal dysfunction was also indicated by changes in proliferation and survival of adult-born dentate gyrus cells in the knockout mice. These results suggest focusing on the functions of hippocampal formation, such as novelty detection, when using the GluA1-/- mouse line as a model for neuropsychiatric and cognitive disorders.